Analyte Detection
For many years, scientists and physicians have sought rapid, simple and reliable methods for isolating and measuring analytes of all kinds including cells, cell organelles, molecules, and atoms. Most methods of isolating and measuring analytes have involved complicated and expensive analytical equipment such as spectrophotometers, densitometers, gamma and scintillation counters, flow cytometric devices, gas and ion exchange chromatographs, affinity columns, high performance liquid chromatographs and the like. In addition, many of these methods involve the use of various kinds of radiolabels such as .sup.125 I, .sup.3 H, .sup.14 C, .sup.32 P and others. These isotopes are expensive, require careful disposal and in some cases pose a health threat to laboratory personnel. Non-radioactive methods of analyte detection involve fluorescent, calorimetric, magnetic, and enzyme-linked assay methods among others. All of these methods involve expensive and sophisticated equipment, multiple additions of various reagents, mixing, incubating at different temperatures, and some separation steps, for example centrifugation, and take several hours if not days to complete. Furthermore, these methods do not permit in vivo measurement of an analyte or measurement of multiple analytes in a single system. Usually, a sample must be removed from a patient and subjected to various processing steps before attempting to include the sample in the assay system.
Accordingly what is needed is a rapid, simple, non-isotopic method that does not involve labeling of analytes or other reagents, and minimizes sample manipulation before performing the assay. Furthermore methods are needed that provide a means to measure one or more than one analyte in vivo and in vitro. What is also needed is a system that learns to detect patterns of analyte values associated with the presence of a certain condition or disease.
Substance Delivery
Health care providers such as physicians, nurses, therapists and others, and research scientists have long sought simple reliable, and controlled methods of delivering drugs and other substances to specific locations. Such targeted substance delivery has often been hampered by the typical problems involving substance solubility and stability, the rate and pattern of release of the substance, dilution in the general circulation before reaching the target site, and toxicity of the substance in systems that are not the desired target site.
What is desirable is a system that provides for targeted substance delivery in a controlled fashion. Such a system should be capable of delivering one or more than one substance, of being implanted into a patient, and of being activated by external signals to release substances at specific times and in specific amounts.
Analyte Detection and Substance Delivery System
What is also needed is a system that detects an analyte, analyzes the amount of analyte, and if the amount exceeds or does not meet a certain threshold, sends a signal to another membrane containing a desired substance for release of the substance at a desired location such as into a patient.
Isolation of Cells and Organelles
The human hematopoietic system is populated by cells of several different lineages. These "blood cells" may appear in bone marrow, the thymus, lymphatic tissue(s) and in blood such as umbilical cord blood, and also arterial blood and venous blood obtained centrally or peripherally. Within any specific lineage, there are a number of maturational stages. In most instances, the more immature developmental stages occur within bone marrow while the more mature and final stages of development occur in peripheral blood.
There are two major lineages: The myeloid lineage which matures into red blood cells, granulocytes, monocytes and megakaryocytes; and the lymphoid lineage which matures into B lymphocytes and T lymphocytes. Within each lineage and between each lineage, antigens are expressed differentially on the surface and in the cytoplasm of the cells in a given lineage. The expression of one or more antigens and/or the intensity of expression can be used to distinguish between maturational stages within a lineage and between lineages. Assignment of cell to lineage and to a maturational stage within a cell lineage indicates lineage commitment.
There are cells, however, which are uncommitted to any lineage (i.e., "progenitor" cells) and which, therefore, retain the ability to differentiate into each lineage. These undifferentiated, pluripotent progenitor cells will hereinafter be referred to as the "stem cells." Therefore, all of mammalian hematopoietic cells can, in theory, be derived from a single stem cell. The stem cell is able to self-renew, so as to maintain a continuous source of pluripotent cells. In addition, when subject to particular environments and/or factors, the stem cells may differentiate to yield dedicated progenitor cells, which in turn may serve as the ancestor cells to a limited number of blood cell types. These ancestor cells will go through a number of stages before ultimately yielding a mature cell. Stem cells may be related to the lympho-hematopoietic system. Other stem cells may be unrelated to the lympho-hematopoietic system and may be derived from ectoderm, mesoderm, and endoderm. These stem cells may include germ cells such as, but not limited to, oogonia and spermatogonia, as well as myoblasts, fibroblasts, osteoblasts and neuroblasts.
The benefit of obtaining a pure population of stem cells is most readily recognized in the field of gene therapy. Briefly, gene therapy can be used to treat specific diseases caused by a defect in a particular gene. For example, sickle cell anemia is caused by a defect in a single gene. The cellular precursors of erythrocytes of sickle cell patients contain this defective gene which, in turn, codes for a defective form of the protein hemoglobin. The defective form results in the clinical condition of sickle cell anemia. Sickle cell anemia cannot be "cured" by conventional drug therapies because the underlying defect is in the gene which is included within every cell. Gene therapy seeks to replace or repopulate the cells of the hematopoietic system with cells that do not contain the defective gene but instead contain a "normal" gene. Using conventional recombinant DNA techniques, a "normal" gene is isolated, placed into a viral vector, and the viral vector is transfected into a cell capable of expressing the product coded for by the gene. The cell then must be introduced into the patient. If the "normal" gene product is produced, the patient is "cured" of the condition.
Bone marrow transplantation is an effective therapy for an increasing number of diseases. Graft Versus Host Disease (GVHD), however, limits bone marrow transplantation to recipients with histocompatibility (HLA)-matched sibling donors. Even then, approximately half of the allogenic bone marrow transplantation recipients develop GVHD. Current therapy for GVHD is imperfect and the disease can be disfiguring and/or lethal. Thus, risk of GVHD restricts the use of bone marrow transplantation to patients with otherwise fatal diseases, such as malignancies, severe aplastic anemia, and congenital immunodeficiency states.
Less than 1000 bone marrow transplantations per year are currently performed in the United States. Many other patients have diseases that might be treated by marrow cell transplantation (such as sickle cell anemia) if GVHD were not such a serious risk. The potential benefits from expanded use of bone marrow transplantation have stimulated research on the cause and prevention of GVHD. It has been shown that donor T lymphocytes cause GVHD in animals. Removal of T lymphocytes from donor marrow inocula ("grafts") prevented the subsequent development of GVHD in mice, dogs and monkeys. Similar trials in humans with monoclonal antibodies against human T lymphocytes are now in progress. Preliminary results, however, suggest only attenuation of GVHD, not a cure. Similar results have been achieved with E-rosette and soybean lectin depletion of T lymphocytes.
Another approach under investigation is the use of anti-T lymphocyte monoclonal antibodies conjugated to toxins, such as ricin. As of yet, however, GVHD has not been prevented or cured in bone marrow recipients. A continuing need exists, therefore, for new methods of collecting stem cells. Donors of bone marrow are also faced with undesirable procedures and risks. The current procedures for harvesting bone marrow are expensive and painful. Furthermore, the current donation procedure is accompanied by the risks associated with anesthesia, analgesia, blood transfusion and possible infection. It would be desirable, therefore, to improve the current method of harvesting marrow from donors.
Selection, Growth and Differentiation of Cells
In addition to the needs for harvesting specific cell types and isolating a wide variety of other analytes, there is also a need to provide specific substrates for cell adhesion, growth and differentiation. Current substrates for attachment and growth of cells in vitro include poly-L-lysine, fibronectin and other molecules. These substrates are primarily passive in nature, and cell growth and differentiation depends on other factors added to the culture medium such as growth factors, cytokines and hormones. These substrates are not capable of being engineered to optimize specific cell adhesion, growth and differentiation. These substrates also lack the capability to select specific cells from a heterogeneous population of cells and to promote the growth and differentiation of these specific cells.
In addition to a need for isolation and controlled differentiation and growth of stem cells that are related to the hematopoietic system, as described above, there is also a need for the isolation, controlled differentiation and growth of stem cells which are not associated with the hematopoietic system. Such stem cells may be derived from ectoderm, mesoderm, and endoderm. These stem cells may include germ cells such as oogonia and spermatogonia, myoblasts, fibroblasts, osteoblasts and neuroblasts.
What is needed are polymer membranes that have characteristic and specific properties for cell adhesion, growth and differentiation. What is also needed are polymer membranes that may be specifically designed and controlled to possess characteristic properties for cell selection, adhesion, growth and differentiation.